Figure 4

Induction of luciferase by tetracycline. (A) Expression of luciferase mRNA in a polyclonal line of tetracycline treated cells. pTcINDEX-Luc transformed epimastigotes were harvested each day following the addition of tetracycline to the cultures (5 μg ml^-1). Lane U is the uninduced cell line. Lanes 1–7 represent days following induction. 1.25 μg total RNA was loaded in each lane. The blot was hybridised with a 1.7 kb Bam HI/Sal I fragment containing the luciferase ORF. (B) Luciferase activity over a time course. Cells were induced and aliquots removed for enzyme assay at indicated timepoints. Dashed line: cells were induced for 24 hours then washed twice in tetracycline-free medium and resuspended in tetracycline-free medium. Solid line: cells were induced with tetracycline then incubated with no further treatment. (C) Effect of tetracycline concentration on the level of luciferase activity. 10 identical flasks of epimastigotes (as above) were treated with different concentrations of tetracycline for 24 hours. Cells were harvested and the luciferase activity measured (Methods). The Y axis indicates the fold increase in luciferase activity above uninduced cells, normalised, to the amount of protein present in each extract.